We have studied stress-induced reversion to virulence of infectious pancreatic necrosis virus (IPNV) in persistently infected Atlantic salmon (Salmo salar L.) fry. Naïve fry were persistently infected with a virulent strain (T217A221 of major structural virus protein 2, VP2) or a low virulent (T217T221) variant of IPNV. The fry were infected prior to immunocompetence as documented by lack of recombination activating gene-1, T-cell receptor and B-cell receptor mRNA expression at time of challenge. The fish were followed over 6 months and monitored monthly for presence of virus and viral genome mutations. No mutation was identified in the TA or TT group over the 6 months period post infection. Six months post infection TA and TT infected groups were subject to daily stress for 7 days and then sampled weekly for an additional period of 28 days post stress. Stress-responses were documented by down-regulation of mRNA expression of IFN-α1 and concomitant increase of replication levels of T217T221 infected fish at day 1 post stress. By 28 days post stress a T221A reversion was found in 3 of 6 fish in the T217T221 infected group. Sequencing of reverted isolates showed single nucleotide peaks on chromatograms for residue 221 for all three isolates and no mix of TA and TT strains. Replication fitness of reverted (TA) and non-reverted (TT) variants was studied in vitro under an antiviral state induced by recombinant IFN-α1. The T217A221 reverted variant replicated to levels 23-fold higher than the T217T221 strain in IFN-α1 treated cells. Finally, reverted TA strains were virulent when tested in an in vivo trial in susceptible salmon fry. In conclusion, these results indicate that stress plays a key role in viral replication in vivo and can facilitate conditions that will allow reversion from attenuated virus variants of IPNV.
In high intensive fish production systems, hyperoxygenation and reduced flow are often used to save water and increase the holding capacity. This commonly used husbandry practice has been shown to be stressful to fish and increase mortality after infectious pancreatic necrosis virus (IPNV) challenge, but the cause and effect relationship is not known. Salmonids are particularly sensitive to stress during smoltification and the first weeks after seawater (SW) transfer. This work aimed at investigating the impact of hyperoxygenation combined with reduced flow in fresh water (FW), on the intestinal barrier in FW as well as during later life stages in SW. It further aims at investigating the role of the intestinal barrier during IPNV challenge and possible secondary infections. Hyperoxygenation in FW acted as a stressor as shown by significantly elevated plasma cortisol levels. This stressful husbandry condition tended to increase paracellular permeability (P(app)) as well as translocation of Aeromonas salmonicida in the posterior intestine of Atlantic salmon. After transfer to SW and subsequent IPNV challenge, intestinal permeability, as shown by P(app), and translocation rate of A. salmonicida increased in the anterior intestine, concomitant with further elevation in plasma cortisol levels. In the anterior intestine, four of five fish displayed alterations in intestinal appearance. In two of five fish, IPNV caused massive necrosis with significant loss of cell material and in a further two fish, IPNV caused increased infiltration of lymphocytes into the epithelium and granulocytes in the lamina propria. Hyperoxygenation and reduced flow in the FW stage may serve as stressors with impact mainly during later stages of development. Fish with an early history of hyperoxygenation showed a higher stress response concomitant with a disturbed intestinal barrier function, which may be a cause for the increased susceptibility to IPNV infection and increased susceptibility to secondary infections.
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